Mechanism Of P-STAT6/Tim-3 In The Regulation Of Tumor Associated Macrophage Polarization And Progression Of Bladder Cancer

BACKGROUNDBladder cancer is one of the most common malignant tumors in urinary system.Nowadays,immunotherapy for tumor microenvironment is a research hotspot in this field,but the mechanism of action has not yet been fully illuminated.Therefore,to reveal tumor microenvironment of bladder cancer in regulating tumor proliferation and invasion and to find new targets in treating bladder cancer are still focuses of bladder cancer research.Tumor associated macrophage(TAM)refers to macrophages infiltrating into cancer tissue and migrating into the tumor stroma.Macrophages mainly have two polarization types:M1 type and M2 type.M1 type is a classical activation type,mainly manifested in pro-inflammatory and anti-tumor activity.M2 type is an alternative activation type,mainly manifested as anti-inflamatory and tumor-promoting activity.TAM is mainly M2 type.The tumor-promotion of TAM may be related to the epithelial-to-mesenchymal transition(EMT)in tumor cells.Recent studies have shown that the expression of signal transducers and activators of transcription 6(STAT6)and T-cell immunoglobulin and mucin domain 3(Tim-3)plays important roles in M2 polarization and the progression of tumor disease.STAT6 has a transcriptional activation function.After activation,phosphorylated STAT6 ·(P-STAT6)will be transferred into the nucleus to initiate target gene expression.Breast cancer studies have shown that STAT6 phosphorylation is closely related to macrophage M2 polarization.AS1517499,STAT6 inhibitor,can block STAT6 phosphorylation and inhibit the growth and metastasis of breast cancer in nude mice.Recent studies have shown that Tim-3 is an inhibitory immune checkpoint molecule,which plays an immunosuppressive function in the tumor microenvironment and induces tumor immune escape.Other studies have shown that Tim-3 expresses highly in TAM and may participate in the polarization of M2 of TAM and promote the progression of tumor diseases,and its expression level is regulated by activation of upstream STAT6 phosphorylation.Previous studies on P-STAT6/Tim-3 showed that phosphorylation of STAT6 in macrophage affected the expression of Tim-3,and the cytokine Interleukin-4(IL-4)was activated by activating monocytes.The transcription factor STAT6,which further binds to the Tim-3 gene promoter,promotes Tim-3 expression.All above studies indicate that M2 polarization of tumor-associated macrophages mediated by P-STAT6/Tim-3 is closely related to immune escape and disease progression of malignant tumors.Studies have shown that the degree of infiltration of M2 type TAM is closely related to poor prognosis of bladder cancer in tumor microenvironment.Our previous study has found that the high expression of STAT6 and Tim-3 in bladder cancer is closely related to the tumor progression,but its specific mechanism of action is still not clear.Whether STAT6 and Tim-3 affect the progression of bladder cancer by mediating the M2 polarization of TAM is not known yet.It was found that AS 1517499.STAT6 inhibitor,could inhibit macrophage M2 polarization by blocking STAT6 phosphorylation.Therefore,we hypothesize that P-STAT6/Tim-3 may induce M2 polarization in tumor associated macrophages in the tumor microenvironment of bladder cancer.Blocking STAT6 phosphorylation in macrophage by AS1517499 may inhibit the proliferation,migration and invasion of bladder cancer,which provides a theoretical basis for the treatment of bladder cancer in the future.OBJECTIVETo investigate the effect of P-STAT6/Tim-3 on the M2 polarization of tumor associated macrophages in bladder cancer.To explore the mechanism of P-STAT6/Tim-3 in regulating and controlling proliferation,migration and invasion of bladder cancer.METHODS1.The role and mechanism of P-STAT6/Tim-3 in the polarization of THP-1 cells into M2 macrophagesTHP-1 cells were induced to MO,M1 and M2 cells with corresponding exogenous cytokines,and the morphological changes of cells were recorded.By Western blotting and flow cytometry,the phenotypes of M0,M1 and M2 were distinguished,the effects of various concentrations of AS1517499 on M1 and M2 polarization were detected,and the expressions of STAT6,P-STAT6 and Tim-3 in M1 and M2 polarization of macrophages were explored.The toxic effects of different concentrations of AS 1517499 on THP-1 cells were detected with CCK-8 cytotoxicity test.2.Effect and mechanism of M2-like polarization induced by bladder cancer cells through P-STAT6/Tim-3.Using macrophage MO and bladder cancer cells T24 and 5637 as the research object.The experimental groups were as follows:MO(control),T24+M0,T24+M0+AS1517499,5637+M0,5637+M0+AS1517499.The concentration of AS1517499 was 100nM.The morphological changes of macrophages in each group were ovserved,and the toxic effects of different concentrations of AS 1517499 on bladder cancer cells were detected with CCK-8 cytotoxicity test.The expressions of CD163,Arg-1,STAT6,P-STAT6 and Tim-3 were detected by Western blotting.The M1 and M2 phenotypes were identified by flow cytometry.The secretion of IL-4,IL-13,IFN-sand TNF-α by bladder cancer cells were detected by using ELISA method.3.Effects of proliferation.invasion and transforming of EMT of bladder cancer through blocking P-STAT6/Tim-3 in macrophage by AS1517499Using macrophage MO and bladder cancer cells T24 and 5637 as the research object.The experimental groups were as follows:T24(T24,T24+M0,T24+M0+AS1517499,T24+AS1517499)and 5637(5637,M0+5637,M0+5637+AS1517499,5637+AS1517499).The concentration of AS1517499 was 100nM.The morphological changes of bladder cancer cells were observed.The proliferation of bladder cancer cells was detected by CCK8 cell proliferation test.The cell migration and invasion of bladder cancer cells were detected by cell scratch test and transwell cell migration/invasion test,and the expressions of E-cadherin,Vimentin,N-cadherin and Ki-67 were detected in bladder cancer cells by Western blotting.4.Effect of AS1517499 on macrophage polarization and tumor growth of bladder cancer cells in nude miceThe experimental models of tumor bearing nude mice were established by bladder cancer cell T24.T24 cells were inoculated into the lateral root of the right leg of 10 male nude mice.The transplanted tumor model was successfully established and divided into 2 groups randomly:the experimental group and the control group,with five rats in each group.The experimental group was given AS 1517499(20mg/kg,two times a week,intraperitoneal injection)and the control group was given equal amount of saline.The nude mice were killed after twenty-eight days.The tumors were removed,photographed and weighed.The expressions of P-STAT6,Tim-3,CD 163 and Ki-67 were detected by using immunofluorescence,immunohistochemistry and Western blotting.5.Expressions and clinical significance of P-STAT6,Tim-3 and CD163 in tumor associated macrophage of bladder cancer53 cases of bladder urothelial carcinoma and 15 cases of normal tissues around cancer specimens were collected.The expressions of P-STAT6,Tim-3,CD 163 and Ki-67 were detected using immunofluorescence and immunohistochemical staining.Analyzing the expressional relationship and clinical significance of P-STAT6,Tim-3 were combined with the pathological grades,clinical stages and Ki-67 expression of bladder cancer patients.6.Statistical analysisAll determinations were made in triplicate.All data were analyzed using SPSS version 17.0.The results were presented as mean ± standard deviation.One and two-way ANOVA-analyses were performed for analyzing statistical differences among groups.The difference between two sets of data was compared by t test or χ2 test.The correlation of data was analyzed using Pearson correlation analysis.P<0.05 was considered to be statistically significant.P<0.01 was considered to be remarkable statistically significant.RESULTS1.The THP-1 cells can be polarized into M2-like macrophages by activating P-STAT6/Tim-3(1)After Ml and M2 polarization,the morphology of THP-1 cells changed from small circle to long shuttle and polygon.(2)The result of Western blotting showed that CD80 and iNOS were highly expressed in M1 group,and CD163,Arg-1,P-STAT6 and Tim-3 were highly expressed in M2 group.(3)The result of flow cytometry showed that CD 80 and IL-6 were highly expressed in M1 group,while CD206 and IL-10 were highly expressed in M2 group.(4)After administration of AS1517499(100nM and 300nM),the expressions of CD 163,Arg-1,P-STAT6 and Tim-3 in M2 group decreased significantly.(5)The result of CCK-8 cytotoxicity test showed that AS1517499(300nM)had significant toxic effect on THP-1 cells.The appropriate drug concentration of AS1517499 is 100nM.2.The bladder cancer cells induced the macrophages polarization into M2 group through the activation of P-STAT6/Tim-3.(1)After co-cultured with bladder cancer cells for 48h,the morphology of THP-1 cells changed from small circle to long shuttle and polygon.(2)The result of CCK-8 cytotoxicity test showed that AS1517499 had no significant toxic effect on bladder cancer cells.(3)The result of Western blotting showed that the expressions of CD163,Arg-1,P-STAT6 and Tim-3 were highly expressed in the co-culture group,and the expressions of CD 163,Arg-1,P-STAT6 and Tim-3 were significantly decreased in co-culture system added with AS1517499(100nM).(4)The result of flow cytometry showed that CD206 and IL-10 were highly expressed in the co-culture group.The expressions of CD206 and IL-10 were significantly decreased in co-culture system added with AS1517499(100nM).(5)The result of ELISA showed that the expressions of IL-4,IL-13,IFN-y and TNF-a in bladder cancer cells were not affected by AS 1517499(1 OOnM).3.The TAM promoted the proliferation,invasion and EMT of bladder cancer cells through P-STAT6/Tim-3 blocked by AS1517499.(1)After co-cultured with MO macrophages for 48h,the morphology of T24 and 5637 of bladder cancer cells changed from approximately circular to long fusiform,and the cell gap became larger.(2)The result of CCK-8 cell proliferation test showed that the proliferation ability of bladder cancer cells in the co-culture group was significantly higher than that in the control group,and the proliferation was restored after adding AS1517499(100nM).(3)The results of cell scratch test and transwell cell migration/invasion test showed that the migration and invasion abilities of bladder cancer cells in the co-culture group was significantly higher than that of the control group.After adding AS1517499(100nM),the migration and invasion abilities were inhibited.(4)The result of Western blotting showed that the expressions of Vimentin,N-cadherin and Ki-67,the related index of EMT in the experimental group,were significantly higher than those in control group.After adding AS 1517499(100nM),the expressions of Vimentin,N-cadherin and Ki-67 decreased.4.The AS1517499 inhibited the growth of bladder cancer in nude mice by blocking the pathway of STAT6/Tim-3(1)The tumor bearing nude mice model of bladder cancer cell T24 was established.The results showed that the volume and weight of the tumors in the AS1517499 group were lower than those of the control group,and the difference was statistically significant.(2)The immunofluorescence staining showed that P-STAT6 and Tim-3 were respectively co-expressed with CD 163,which represented the expression of P-STAT6 and Tim-3 in M2 type macrophages respectively.The results of immunohistochemistry and Western blotting showed that the expressions of Ki-67,P-STAT6,Tim-3 and CD163 in tumor tissues were all lower than those in the control group,and the difference was statistically significant.5.The expressions of P-STAT6 and Tim-3 in bladder cancer cells were closely related to the pathological grades and clinical stages of bladder cancer.(1)The immunofluorescence showed that P-STAT6 and Tim-3 were significantly co-expressed with CD163,which represented the expressions of P-STAT6 and Tim-3 in M2 type macrophages respectively.(2)The expressions of P-STAT6,Tim-3 and CD 163 in TAM were closely related each other and were significantly higher than those in normal bladder mucosa.(3)The analysis of the clinical and pathological data showed that the expressions of P-STAT6,Tim-3 in TAM were closely related to the pathological grades,clinical stages and Ki-67 expression of bladder cancer.CONCLUSIONS(1)Bladder cancer cells and exogenous cytokines could induce M2-like polarization of TAM by activating P-STAT6/Tim-3 in macrophages.The M2 type TAM could further promote EMT,proliferation,migration and invasion of bladder cancer cells.(2)AS 1517499 could inhibit the M2-like polarization of TAM by blocking the P-STAT6/Tim-3 of macrophage,and then inhibit proliferation,migration,invasion and EMT of bladder cancer cells.(3)The expressions of P-STAT6 and Tim-3 in tumor associated macrophages were consistent and correlated with pathological grades,clinical stages and Ki-67 expression of bladder cancer.